The long-term goal of this project is to study how post-translational modifications of DNA mismatch repair (MMR) proteins impact genome integrity and cancer development. MMR maintains genome stability by removing mismatches in the newly synthesized strand during DNA replication. The MMR reaction involves mismatch recognition by the initiation factors (MutS, MutL, and PCNA), mismatch removal by nucleases, and DNA gap-filling by DNA polymerases. The importance of MMR is under- scored by the fact that MMR defects lead to hypermutations and susceptibility to both hereditary and sporadic colorectal cancers (CRCs). Exhibiting elevated instability in simple repeats, called microsatellite instability (MSI), is a hallmark of MMR-deficient CRCs. However, only ~70% of hereditary and sporadic CRC cases that display MSI have identifiable mutations in MMR genes, suggesting that other mechanism(s) are responsible for the MSI phenotype in the remaining 30% of the cases. We recently showed that CRC cells containing high levels of PCNA tyrosine phosphorylation are defective in MMR in vitro. We therefore hypothesize that PCNA phosphorylation inhibits MMR, leading to genome instability and CRC development. To test this hypothesis, three Specific Aims are proposed. Aim 1 is to determine how phosphorylated PCNA inhibits MMR. A well-defined in vitro MMR reaction will be conducted in both a nuclear extract system and a reconstituted system in the presence or absence of phosphorylated PCNA, and analysis of the repair products will allow determination of the specific step(s) of the reaction tht is blocked. Aim 2 is to determine hypermutability and MMR proficiency in cells stably expressing phosphorylated PCNA mimics while suppressed for endogenous PCNA expression. Aim 3 is to analyze tumorigenesis in transgenic mice expressing phosphorylated PCNA. A successful completion of the proposed work will establish PCNA tyrosine phosphorylation as a novel biomarker for cancer etiology and progression.